scholarly journals NTN1 Affects Porcine Intramuscular Fat Content by Affecting the Expression of Myogenic Regulatory Factors

Animals ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 609 ◽  
Author(s):  
Ligang Wang ◽  
Lingling Zhao ◽  
Longchao Zhang ◽  
Xin Liu ◽  
Xinhua Hou ◽  
...  

Intramuscular fat (IMF) content is an important economic trait for pork quality. Our previous results regarding the genome-wide association between IMF content and copy number variations (CNVs) indicated that the CNV within Netrin-1(NTN1-CNV) was significantly associated with IMF. In order to validate the effect of NTN1-CNV, we detected the Netrin-1 (NTN1) gene dose and protein expression content in the longissimus dorsi of different IMF content pigs using Western blotting and investigated the expression of NTN1 RNA in different tissues using real-time quantitative polymerase chain reaction (qPCR). The knock-down of the NTN1 gene in C2C12 and 3T3-L1 cells and over-expression in C2C12 cells during the proliferation and differentiation stage were also investigated to explore the possible pathway of action of NTN1. The results showed that in individuals with IMF content differences, the gene dose of NTN1 and the expression of NTN1 protein were also significantly different, which indicated that NTN1-CNV may directly affect IMF by its coding protein. NTN1 had the highest expression in pig longissimus dorsi and backfat tissues, which indicates that NTN1 may play an important role in muscle and fat tissues. The in vitro validation assay indicated that NTN1 silencing could promote the proliferation and inhibit the differentiation of C2C12 cells, with no effect on 3T3-L1 cells. Additionally, NTN1 over-expression could inhibit the proliferation and promote the differentiation of C2C12 cells. Combined with previous research, we conclude that NTN1-CNV may affect IMF by its gene dose, and the expression of NTN1 may affect the proliferation and differentiation of muscle cells by the AMP-activated protein kinase (AMPK) pathway and finally influence the IMF.

Genes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 81
Author(s):  
Natalia Leciejewska ◽  
Ewa Pruszyńska-Oszmałek ◽  
Karolina Mielnik ◽  
Maciej Głowacki ◽  
Tomasz P. Lehmann ◽  
...  

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.


2020 ◽  
Author(s):  
Ruo-nan Zhang ◽  
Yan Wang ◽  
Yun Liu ◽  
Xin Bao ◽  
Wei Xu ◽  
...  

Abstract Backgorund:Neuromuscular diseases are a kind of nervous system diseases that have a high disability rate.Ezrin’ role in skeletal muscle has not been identified. This study aims to confirm the effect and mechanism of Ezrin on myoblast differentiation and fusion, myotube size, and myofiber type.Method:By using immunoassaying and western blot analyses, Ezrin, MyHC,MEF2c, MyoG, PKAα/β/γ, PKA reg Iα, PKA reg IIβand NFATc1-c4 were detected in myoblast cells treated with Ad-Ezrin or Ad-shEzrin. Real-time PCR were used to evaluate MyoD, Myf5, MyHC-I , MyHC-IIa/b and MyHC-IIx in myoblast cells. PKA inhibitor H-89 or PKAreg I activator N6-Bz-cAMP were added into medium to confirm their relationship between Ezrin and PKA during myoblast differentiation/fusion. In vitro, Ad-NFATc1/c2 or Ad-shNFATc3/c4 were respectively transfected into C2C12 cells, myoblast differentiation/fusion, myotube size and myofiber type were assessed by using immunostaining of MyHC, MEF2c and MyoG. In vivo, transfection of Ad-Ezrin into gastrocnemius and soleus muscles for 7 days, the numbers of MyHC-1 postivemyofibers were analyzed after immunostaining of MyHC-1.Results: Ezrin expression were time-dependently increased during myoblast differentiation/fusion. Knockdown of Ezrin by shRNA delayed myoblast differentiation and fusion in a time dose-dependent pattern, as shown by immunostaining of MyHC. Conversely, over-expression of Ezrin by adenovirus time- and dosage-dependently promoted myoblastdifferentiation/fusion, and muscle fiber specialization characterized by increased MyHC I and MyHCIIa/b. Forced expression of Ezrin did not alter PKA, and PKAreg II α levels, but altered the levels of PKAreg I α/β, Myf5 and MyoD, and leading to the accumulation of MyoG+/MEF2c+ nuclei. By contrast, Ezrin knockdown significantly decreased the PKA reg I/II ratio and MyoG+/MEF2c+ nuclei. The PKA inhibitor H-89 remarkably abolished the beneficial effect of over-expressingEzrin on the numbers of MyHC+ myotubes and MyoG+/MEF2c nuclei. These opposite changes mediated by knocking down Ezrin were almost eliminated by PKAreg I activator N6-Bz-cAMP. Furthermore, over-expression of NFATc2 or knockdown of NFATc4reversed the inhibitory effect of Ezrin knockdown on myoblast differentiation/fusion, resulting in the recovery of the numbers ofMyoG+/MEF2c+ nucleiin3-nuclei+myotubes. Meanwhile, overexpression of Ezrin specifically induced type I muscle fiber specialization, which was associated with increased levels of NFATc1/c2. Furthermore, in vivo transfection ofAd-Ezrin into gastrocnemius and soleus muscles increased the numbers of MyHC-1 postivemyofibers. By contrast, knockdown of NFATc4resulted in the recovery to normal levels of MyHC-2b in Ezrin-knockdown myoblast cells, attributingtoregainingMyoDand MEF2c expression. Conclusions: Ezrin trigger myoblast differentiation and fusion, myotube size, and alters muscle fiber specialization through PKA-NFAT-MyoD/MEF2C signalling pathway.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwang Zhang ◽  
Tingli Pan ◽  
Yu Sun ◽  
Siqi Liu ◽  
Ziyi Song ◽  
...  

Abstract Background In the livestock industry, intramuscular fat content is a key factor affecting meat quality. Many studies have shown that dietary calcium supplementation is closely related to lipid metabolism. However, few studies have examined the relationship between dietary calcium supplementation and intramuscular fat accumulation. Methods Here, we used C2C12 cells, C57BL/6 mice (n = 8) and three-way cross-breeding pigs (Duroc×Landrace×Large white) (n = 10) to study the effect of calcium addition on intramuscular fat accumulation. In vitro, we used calcium chloride to adjust the calcium levels in the medium (2 mmol/L or 3 mmol/L). Then we measured various indicators. In vivo, calcium carbonate was used to regulate calcium levels in feeds (Mice: 0.5% calcium or 1.2% calcium) (Pigs: 0.9% calcium or 1.5% calcium). Then we tested the mice gastrocnemius muscle triglyceride content, pig longissimus dorsi muscle meat quality and lipidomics. Results In vitro, calcium addition (3 mmol/L) had no significant effect on cell proliferation, but promoted the differentiation of C2C12 cells into slow-twitch fibers. Calcium supplementation increased triglyceride accumulation in C2C12 cells. Calcium addition increased the number of mitochondria and also increased the calcium level in the mitochondria and reduced the of key enzymes activity involved in β-oxidation such as acyl-coenzyme A dehydrogenase. Decreasing mitochondrial calcium level can alleviate lipid accumulation induced by calcium addition. In addition, calcium addition also reduced the glycolytic capacity and glycolytic conversion rate of C2C12 cells. In vivo, dietary calcium supplementation (1.2%) promoted the accumulation of triglycerides in the gastrocnemius muscle of mice. Dietary calcium supplementation (1.5%) had no effect on pig weight, but significantly improved the flesh color of the longissimus dorsi muscle, reduced the backfat thickness and increased intramuscular fat content in pigs. Besides, calcium addition had no effect on longissimus dorsi pH, electrical conductivity and shear force. Conclusions These results suggest that calcium addition promotes intramuscular fat accumulation by inhibiting the oxidation of fatty acids. These findings provide a new tool for increasing intramuscular fat content and an economical strategy for improving meat quality.


2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Di Xiao ◽  
Ruiye Bi ◽  
Xianwen Liu ◽  
Jie Mei ◽  
Nan Jiang ◽  
...  

Abstract Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment. In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the ‘regulatory sensitivity’ of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn’t significant changes of this ‘regulatory sensitivity’ of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the ‘regulatory sensitivity’ of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Constanze Kröber-Boncardo ◽  
Stephan Lorenzen ◽  
Christine Brinker ◽  
Joachim Clos

Abstract Leishmania donovani is a trypanosomatidic parasite and causes the lethal kala-azar fever, a neglected tropical disease. The Trypanosomatida are devoid of transcriptional gene regulation and rely on gene copy number variations and translational control for their adaption to changing conditions. To survive at mammalian tissue temperatures, L. donovani relies on the small heat shock protein HSP23, the loss of which renders the parasites stress sensitive and impairs their proliferation. Here, we analysed a spontaneous escape mutant with wild type-like in vitro growth. Further selection of this escape strains resulted in a complete reversion of the phenotype. Whole genome sequencing revealed a correlation between stress tolerance and the massive amplification of a six-gene cluster on chromosome 35, with further analysis showing over expression of the casein kinase 1.2 gene as responsible. In vitro phosphorylation experiments established both HSP23 and the related P23 co-chaperone as substrates and modulators of casein kinase 1.2, providing evidence for another crucial link between chaperones and signal transduction protein kinases in this early branching eukaryote.


2012 ◽  
Vol 25 (1) ◽  
pp. 239-246 ◽  
Author(s):  
E. Nucera ◽  
C. Nicoletti ◽  
C. Chiapparino ◽  
M.L. Pacello ◽  
V. D'Alessio ◽  
...  

AvidinOX® a product containing aldehyde groups, generated by ligand-assisted sugar oxidation of avidin by sodium periodate, maintains the capacity to bind biotin with very high affinity and exhibits the property to chemically link cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks. In recent studies, we have shown that AvidinOX exhibits much higher persistency in the skeletal muscle than native avidin. The aim of the present study is to evaluate whether AvidinOX-biotin interaction might be exploited to target biotinylated cells to an AvidinOX pre-treated muscle. To accomplish this we performed the following experiments: 1) The proliferation and differentiation properties of biotinylated C2C12 myoblasts were tested in vitro upon linkage to AvidinOX; 2) Bone marrow-derived cells (BMDC) were isolated from GFP positive transgenic mice [strain C57 BL/6-tg (UBC-GFP)] and after biotinylation (bBMDC) were intravenously administered to naive and MAVA+ (Mouse anti Avidin Antibody) C57/B6 mice previously injected with AvidinOX in a tibial muscle (TM). Localization efficiency of GFP+ bBMDC was evaluated on serial sections of the AvidinOX- and vehicle-treated (contra lateral limb) TM, 5 days after transplantation. Results show that biotinylated C2C12 cells, once linked to AvidinOX, maintain their proliferation and differentiation capacity, in vitro. Intravenous injection of biotinylated GFP+ bone marrow-derived cells leads to their specific and efficient localization in the AvidinOX-pre-treated, but not contra lateral muscle of both naive and MAVA+ mice. The present data suggest a potential use of AvidinOX to improve tissue targeted delivery of biotinylated cells.


2001 ◽  
Vol 21 (24) ◽  
pp. 8461-8470 ◽  
Author(s):  
Giulia Fontemaggi ◽  
Aymone Gurtner ◽  
Sabrina Strano ◽  
Yujiro Higashi ◽  
Ada Sacchi ◽  
...  

ABSTRACT The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53+/+ and p53−/− cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73−/−-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.


2021 ◽  
Author(s):  
Rong Wan ◽  
Qingxiang Meng ◽  
Zhou Zhenming ◽  
Wu hao

ABSTRACTConjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has previously been shown to be able to increase intramuscular fat content in vivo and stimulate adipogenesis in intramuscular preadipocytes in vitro in pig. Unfortunately, there is little data to evaluate the effect of CLA on proliferation and differentiation of bovine intramuscular preadipocytes. This study investigated the regulation by CLA in proliferation and differentiation of bovine intramuscular preadipocytes. The results demonstrated that CLA significantly induced the expression of PPARγ and C/EBPα mRNA of bovine intramuscular preadipocytes as well as the accumulation of lipid in cultured intramuscular preadipocytes. Additionally, CLA significantly decreased the cell proportion of phase G0/G1, and remarkably increased the proportion of phase S+G2/M. Collectively, these results suggest that CLA promotes bovine intramuscular preadipocyte proliferation and differentiation.


2013 ◽  
Vol 58 (No. 8) ◽  
pp. 381-388 ◽  
Author(s):  
X. Tao ◽  
X.M. Men ◽  
B. Deng ◽  
Z.W. Xu

The effects of breed, development, and nutrition on mRNA expression of the fat mass and obesity-associated gene (FTO) and its relationship with intramuscular fat (IMF) content in porcine muscle (m. longissimus dorsi; m.l.d.) were estimated. Purebred Jinhua, Zhongbai, Yorkshire, Duroc, Duroc &times; Zhongbai (DZ), and Duroc &times; Yorkshire &times; Landrace (DYL) pigs were used to investigate the effect of breed. Pigs weighing 2.5, 10, 20, 40, 60, and 100 kg were selected to study the effects of different stages of development. To study the effect of nutrition, four diets were selected: corn-soybean (CS), CS with 1.2% conjugated linoleic acid (CLA) or 0.05% creatine monohydrate (CMH), and barley-soybean (BS). All eighty animals were slaughtered, and m.l.d. samples were collected to examine FTO mRNA expression and IMF content. Results showed that breed significantly affected FTO mRNA expression and IMF content. FTO mRNA expression in the studied pigs was in the order: Zhongbai and Yorkshire &gt; Duroc and DZ &gt; Jinhua and DYL. The IMF content ordered by breed was Duroc &gt; DZ &gt; DYL &gt; Jinhua &gt; Zhongbai&nbsp;&gt; Yorkshire. Both FTO mRNA expression and IMF content increased with age of the pigs, with the greatest difference seen between 100 kg pigs and all other weights. In the study, none of the four diets had a significant effect (P &gt; 0.05) on FTO mRNA expression or IMF content. The study demonstrated that FTO mRNA expression increased with increasing body weight and was significantly affected by the breed of pigs. The results showed that FTO mRNA expression had an inconsistent correlation with IMF content between breeds and developmental ages. &nbsp; &nbsp;


2005 ◽  
Vol 13 (2) ◽  
pp. 77-85 ◽  
Author(s):  
M. Prevolnik ◽  
M. Čandek-Potokar ◽  
D. Škorjanc ◽  
Š. Velikonja-Bolta ◽  
M. Škrlep ◽  
...  

Prediction ability of near infrared (NIR) spectroscopy for intramuscular fat content (IMF) determination was studied. The material comprised 126 muscle samples; 46 pig longissimus dorsi and semitendinosus and 34 beef longissimus dorsi muscle samples. The IMF content was chemically determined in duplicate using two different chemical methods; fat extraction according to Folch et al. and Soxhlet extraction with hydrolysis according to SIST ISO 1443. Folch extraction underestimated IMF content compared to Soxhlet extraction with hydrolysis (-0.32%, P < 0.0001). Similar repeatability was obtained for Folch and Soxhlet extraction with hydrolysis (0.17% and 0.18%, respectively, P < 0.0001). Sample spectra were scanned from 400–2500 nm by the NIR Systems model 6500 spectrophotometer (Silver Spring, MD, USA) and analysed by WinISI II on minced and intact (pork only) samples. Modified partial least squares regression was used to develop models and to obtain calibration statistics: coefficient of determination in calibration( R2 C) and cross-validation ( R2 CV) and standard error in calibration ( SEC) and cross-validation ( SECV). We prepared different models (for a single muscle/common, by applying NIR spectrum or the whole spectrum, on intact and minced samples). Obtained models proved the remarkable prediction ability of NIR spectroscopy to determine IMF content ( R2 CV between 0.84 and 0.99; SECV between 0.14% and 0.53%) and confirms the potential of NIR spectroscopy to replace laborious chemical procedures. Regarding the factors studied, calibrations were less accurate for intact than for minced samples; the use of an NIR spectrum compared to the whole spectrum had no important effect on the prediction ability. According to calibration statistics, the prediction using a common equation for several muscles seems more reliable than the equations within the muscle, but the latter showed lower bias.


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